Ferredoxin Reductase Is Critical for P53-Dependent Tumor Suppression Via Iron Regulatory Protein 2

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Ferredoxin reductase is critical for p53- dependent tumor suppression via iron regulatory protein 2

Yanhong Zhang,1,9 Yingjuan Qian,1,2,9 Jin Zhang,1 Wensheng Yan,1 Yong-Sam Jung,1,2 Mingyi Chen,3 Eric Huang,4 Kent Lloyd,5 Yuyou Duan,6 Jian Wang,7 Gang Liu,8 and Xinbin Chen1 1Comparative Oncology Laboratory, Schools of Veterinary Medicine and Medicine, University of California at Davis, Davis, California 95616, USA; 2College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210014, China; 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 4Department of Pathology, School of Medicine, University of California at Davis Health, Sacramento, California 95817, USA; 5Department of Surgery, School of Medicine, University of California at Davis Health, Sacramento, California 95817, USA; 6Department of Dermatology and Internal Medicine, University of California at Davis Health, Sacramento, California 95616, USA; 7Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201 USA; 8Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Ferredoxin reductase (FDXR), a target of p53, modulates p53-dependent apoptosis and is necessary for steroidogenesis and biogenesis of iron–sulfur clusters. To determine the biological function of FDXR, we generated a Fdxr- deficient mouse model and found that loss of Fdxr led to embryonic lethality potentially due to iron overload in developing embryos. Interestingly, mice heterozygous in Fdxr had a short life span and were prone to spontaneous tumors and liver abnormalities, including steatosis, hepatitis, and hepatocellular carcinoma. We also found that FDXR was necessary for mitochondrial iron homeostasis and proper expression of several master regulators of iron metabolism, including iron regulatory protein 2 (IRP2). Surprisingly, we found that p53 mRNA translation was suppressed by FDXR deficiency via IRP2. Moreover, we found that the signal from FDXR to iron homeostasis and the p53 pathway was transduced by ferredoxin 2, a substrate of FDXR. Finally, we found that p53 played a role in iron homeostasis and was required for FDXR-mediated iron metabolism. Together, we conclude that FDXR and p53 are mutually regulated and that the FDXR–p53 loop is critical for tumor suppression via iron homeostasis. [Keywords: FDXR; p53; FDX1; FDX2; IRP2; iron homeostasis; mRNA translation] Supplemental material is available for this article. Received March 24, 2017; revised version accepted June 26, 2017.

The p53 tumor suppressor is a transcription factor and can pecially cancer (Vogelstein et al. 2000; McLure et al. 2004; be activated in response to an array of stresses, such as Yang et al. 2006; Kawauchi et al. 2008; Puzio-Kuter 2011; DNA damage (Nelson and Kastan 1994), oncogene activa- Shen et al. 2014; Funauchi et al. 2015). tion (Lowe and Ruley 1993), and hypoxia (Graeber et al. Iron is essential for a variety of cellular and biochemical 1996). p53 transcriptional activity is also modulated by activities, including energy production and biogenesis of cellular redox state and the abundance of ADP and iron iron–sulfur (Fe-S) clusters (Hentze et al. 2010; Wang and in response to altered energy and iron metabolism (Vogel- Pantopoulos 2011; Lawen and Lane 2013). On the other stein et al. 2000; Abeysinghe et al. 2001; Liang and Rich- hand, an excess amount of iron is known to cause an array ardson 2003; Whitnall et al. 2006; Yang et al. 2006; of abnormalities (Hentze et al. 2010; Wang and Panto- Kawauchi et al. 2008; Dongiovanni et al. 2010; Puzio- poulos 2011; Lawen and Lane 2013). The role of iron Kuter 2011; Shen et al. 2014). Upon activation, p53 induc- overload in cancer development is evidenced by the fact es a plethora of genes for prosurvival (such as p21), proa- that patients suffering from hereditary hemochromato- poptotic (such as Puma), and many other biological sis, a genetic disease characterized by morbid iron responses (Riley et al. 2008; Vousden and Prives 2009). p53 also regulates several aspects of cellular metabolism critical for development and various disease processes, es- © 2017 Zhang et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- 9These authors contributed equally to this work. cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http://creati- online at http://www.genesdev.org/cgi/doi/10.1101/gad.299388.117. vecommons.org/licenses/by-nc/4.0/.

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Zhang et al. accumulation, are prone to hepatocellular carcinoma Results (HCC) and colorectal carcinoma (Shaheen et al. 2003; Beu- tler 2006; Simcox and McClain 2013). Additionally, high Fdxr is essential for embryonic development intake of dietary iron is associated with an increased risk To explore the biological function of FDXR, we generated of cancer (Nishina et al. 2008; Pietrangelo 2009; Sorrentino Fdxr-deficient mice in which the Fdxr gene was deleted et al. 2009). Thus, proper control of iron homeostasis is crit- through homologous recombination (Supplemental Fig. ical for suppressing tumorigenesis. Systemic iron homeo- S1A,B). We found that Fdxr+/− mice were healthy and fer- stasis is controlled primarily by hepcidin, a peptide tile and did not exhibit obvious abnormality within the hormone secreted from the liver (Nemeth et al. 2004). first 6 mo. However, no single Fdxr−/− mouse was found However, cellular iron homeostasis is controlled primarily among 204 newborn animals from intercrosses of Fdxr+/− by iron regulatory protein 1 (IRP1) and IRP2 (Hentze et al. mice (Fig. 1A), suggesting that complete loss of Fdxr in- 1989; Rouault et al. 1990). As an RNA-binding protein, duces embryonic lethality. Thus, we isolated embryos at IRP1/2 regulates gene expression through mRNA stability various developmental stages and showed that Fdxr−/− and/or translation, including transferrin receptor 1 (TfR1) embryos were alive at embryonic days 7.0–8.0 (E7.0– and ferritin heavy chain 1 (FTH1) (Butt et al. 1996; Hender- E8.0) but resorbed at E8.5–E13.5 (Fig. 1A; Supplemental son et al. 1996; Rouault 2006). Interestingly, iron homeo- Fig. S1C). We also found that E8.0 Fdxr−/− embryos exhib- stasis is also modulated by the p53 pathway potentially ited severe developmental defects, whereas E7.5 Fdxr−/− via post-transcriptional regulation of TfR1 and FTH1 ex- embryos failed to develop normal layers of embryonic pression (Zhang et al. 2008). and extraembryonic tissues, including visceral endoderm, Ferredoxin reductase (FDXR), a mitochondrial flavopro- mesoderm, embryonic ectoderm, extraembryonic ecto- tein, transfers an electron from NADPH to ferredoxin derm, and extraembryonic visceral endoderm (Supple- 1 (FDX1) and FDX2 and then to cytochrome P450 for ster- mental Fig. S1D). oidogenesis and biogenesis of Fe-S clusters and heme Since FDXR is necessary for biogenesis of Fe-S clusters A (Brandt and Vickery 1992; Lange et al. 2000; Muller and is a critical modulator of cellular iron homeostasis et al. 2001; Sheftel et al. 2010; Shi et al. 2012). We and (Lange et al. 2000; Sheftel et al. 2010; Shi et al. 2012), we others found that FDXR is a p53 target and sensitizes tu- examined whether embryonic lethality is associated mor cells to 5-fluorouracil- and H O -induced apoptosis 2 2 with aberrant iron metabolism in Fdxr−/− embryos. (Hwang et al. 2001; Liu and Chen 2002). Through interac- Thus, Pearl’s Prussian blue staining was performed and tion with the Fhit tumor suppressor, FDXR modulates ap- showed that iron accumulation was extensive in Fdxr−/− optosis induced by Fhit (Trapasso et al. 2008). Through embryos, moderate in Fdxr+/− embryos, and sparse in interaction with MPZL, a mitochondrial protein and reac- wild-type embryos (Fig. 1B). Thus, aberrant iron accumu- tive oxygen species (ROS) inducer, FDXR regulates epider- lation in developing Fdxr−/− embryos would generate re- mal cell differentiation via mitochondria (Bhaduri et al. active radicals and oxidative stress, leading to 2015). FDXR is also found to be the most consistent inter- embryonic lethality at E8.5 (Fig. 1A), which is similar to nal biodosimetry marker in the peripheral blood following what was observed in FBXL5−/− embryos that died at radiation therapy (Abend et al. 2016; Edmondson et al. E8.5 (Moroishi et al. 2011; Ruiz et al. 2013). 2016). Moreover, FDXR is a potential marker for efficacy of chemotherapy (Yu et al. 2003; Okumura et al. 2015). These studies indicate that FDXR has multiple cellular Mice deficient in Fdxr are prone to spontaneous tumors and biochemical activities, but its biological function and liver abnormalities has not been explored in vivo. Using both FDXR-deficient cell lines and mouse models, we made novel observations A cohort of wild-type (n = 32) and Fdxr+/− (n = 31) mice was that FDXR and p53 are mutually regulated and that the generated to examine median survival, tumor incidence, FDXR–p53 loop is critical for tumor suppression via iron and other abnormalities (Supplemental Tables S1, S2). homeostasis. We found that the median survival for Fdxr+/− mice (102

Figure 1. Loss of Fdxr leads to embryonic lethality potentially due to iron accumulation. (A) The number and percentage of embryos and live offspring from the intercrosses of Fdxr+/− mice. (B) Fdxr deficiency leads to iron overload in E8.0 embryos. Wild-type, Fdxr+/−, and Fdxr−/− embryos were stained with Prussian blue. The right panels represent higher magnification of the boxed regions in the left panels of each set. Blue staining represents iron accumulation.

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The p53–FDXR loop in tumor suppression wk) was significantly shorter than that for wild-type mice S2C). In contrast, Fdxr+/− livers had decreased reticulin (117 wk; P < 0.001 by log rank test) (Fig. 2A). We also found staining along with widened trabeculae, encompassing that Fdxr+/− mice were prone to a broad spectrum of spon- three or more cell layers (Fig. 2F; Supplemental Fig. taneous tumors, especially high-grade malignant sarcoma S2C). Additionally, we found that the transcript for α-feto- (seven out of 29 mice), lung carcinoma (four out of 29 protein (AFP), an HCC biomarker (Farinati et al. 2006), mice), liver tumors (11 out of 29 mice; two HCCs out of was highly induced in Fdxr+/− livers compared with that 11 liver tumors), and high-grade lymphoma (19 out of 29 in wild-type livers (Fig. 2G). Since aberrant iron metabo- mice) (Fig. 2B–F; Supplemental Fig. S2A–I; Supplemental lism leads to liver abnormalities (Andrews 1999; Fleming Tables S1, S2). and Ponka 2012; Simcox and McClain 2013), Pearl’s Among 29 Fdxr+/− mice whose organs were available for Prussian blue staining was performed and showed that histological analysis, 15 had steatosis, 10 had chronic hep- iron accumulation was extensive in Fdxr+/− hepatocytes atitis, and 11 had liver tumors (Fig. 2D; Supplemental Ta- compared with that in wild-type hepatocytes (Fig. 2H; ble S2). Fisher’s exact test showed that the difference Supplemental Fig. S2D). Since mutant p53 is known to between wild-type and Fdxr+/− mice is significant in liver promote HCC development, RT–PCR was performed to steatosis (P = 0.0014), hepatitis (P = 0.0212), and liver tu- determine the p53 status in these 11 Fdxr+/− liver tumor mors (P = 0.0304). Since reticulin staining is used to differ- tissues. No mutation was detected in the p53 DNA-bind- entiate HCC from normal livers (Bergman et al. 1997), ing domain, where the vast majority of mutations occurs, both wild-type and Fdxr+/− livers were stained for reticu- suggesting that p53 mutation is likely rare in Fdxr-defi- lin. We found that wild-type livers showed a well-pre- cient cells and may not play a role in tumorigenesis in- served reticulin network (Fig. 2F; Supplemental Fig. duced by Fdxr deficiency.

Figure 2. Mice deficient in Fdxr are prone to spontaneous tumors. (A) Kaplan-Meier survival curve for wild-type and Fdxr+/− mice. (B) Mice deficient in Fdxr are prone to spontaneous tumors. Tumor spectrum and penetrance in a cohort of wild-type and Fdxr+/− mice are shown. (C) The numbers and percentages of wild-type and Fdxr+/− mice with sarcoma and lymphoma. (D) The numbers and percentages of wild-type and Fdxr+/− mice with liver steatosis, hepatitis, and liver tumors. (E) Representative images of hematoxylin and eosin (H&E)-stained lung tumors in Fdxr+/− mice. (F) Representa- tive images of a normal liver from a wild- type mouse and a liver tumor from a Fdxr+/ − mouse that were stained with H&E(left panel of each set) or reticulin (right panel of each set). (G) The level of α-fetoprotein (AFP) transcript in liver tissues is increased by Fdxr deficiency. The levels of AFP and actin transcripts were measured in wild-type and Fdxr+/− mouse livers. (H) Fdxr deficiency leads to iron overload in liver tissues. Livers from wild-type and Fdxr+/− mice were stained with Prussian blue. Blue staining represents iron accumulation in Fdxr+/− hepatocytes.

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Zhang et al.

Iron metabolism and the p53 pathway are regulated by cient liver tissues, IRP2 and TfR1 were increased, whereas FDXR deficiency IRP1 and FTH1 were decreased by Fdxr deficiency in MEFs (Fig. 3B). To determine what pathway is responsible for aberrant To explore how FDXR deficiency modulates iron me- iron accumulation in developing embryos, several master tabolism, CRISPR–cas9 was used to generate multiple regulators for iron metabolism were examined in wild- FDXR-deficient HCT116 cell lines. Interestingly, the type and Fdxr+/− littermate liver tissues. We found that HCT116 cell line carries three copies of the FDXR gene Fdxr deficiency led to increased expression of IRP2 and (Masramon et al. 2000; Hwang et al. 2001). We were able TfR1 but decreased expression of IRP1 and FTH1 (Fig. to generate multiple clones in which two of the three 3A; Supplemental Fig. S3A). We also found that knock- FDXR alleles were deleted. However, we were unable to down of FDXR in HepG2 and HCT116 cells led to similar generate a single clone in which all three alleles were regulation for IRP1/2, TfR1, and FTH1 (Supplemental Fig. knocked out. These observations are consistent with a S3B,C). Next, the effect of Fdxr deficiency on iron metab- previous report that FDXR is necessary for cell survival olism was examined in wild-type and Fdxr-deficient (Hwang et al. 2001) as well as with the above observation mouse embryonic fibroblasts (MEFs). Due to the require- that Fdxr is necessary for embryonic development (Fig. 1). ment of FDXR for embryonic development (Fig. 1), only Two FDXR+/−/− HCT116 clones (clones #4 and #5) were Fdxr+/− MEFs were generated (Fig. 3B). As in Fdxr-defi- selected for further studies. Sequence analysis showed

Figure 3. Iron metabolism and the p53 pathway are regulated by FDXR. (A–C) West- ern blots were prepared using extracts from wild-type and Fdxr+/− mouse livers (A) and MEFs (B) and wild-type and FDXR+/−/− HCT116 cells (C). The blots were probed with antibodies against FDXR, IRP1, IRP2, TfR1, FTH1, p53, p21, and actin, respectively. (D,E) FDXR deficiency leads to mitochondrial iron accumulation. The level of cytosolic and mitochondrial iron (Fe2+)was measured by QuantiChrom iron assay in a pair of wild-type and Fdxr+/− littermate MEFs (D) and in isogenic control and FDXR+/−/− HCT116 cells (E). The level of cytosolic iron in wild-type MEFs and isogenic control HCT116 cells (the first left column) was set at 1.0. Data are mean ± SD from three independent experiments. (F) Ectopic expression of FDXR restores near-normal expression of iron regulatory proteins and p53. Isogenic control and FDXR+/−/− HCT116 cells were transfected with 1 µg of control pcDNA3 (−) or a vector expressing FDXR with an HA tag at its C terminus (FDXR-HA) (+) for 24 h followed by Western blot analysis with various antibodies as indicated. (G) Ectopic expression of FDXR abro- gates mitochondrial iron overload in FDXR- deficient cells. The experiment was performed as in F. The level of cytosolic and mitochondrial iron (Fe2+) was measured in isogenic control and FDXR+/−/− HCT116 cells transfected with control pcDNA3 (−) or a vector expressing FDXR-HA (+). (First left column) The level of cytosolic iron in isogenic control HCT116 cells transfected with control pcDNA3 was set at 1.0. Data are mean ± SD from three independent experiments.

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The p53–FDXR loop in tumor suppression that allele #1 had no deletion or mutation, allele #2 had a HCT116 cells (Supplemental Fig. S4M–O). These data 20-nucleotide (nt) out-of-frame deletion in exon 9, and al- suggest that FDXR may play a role in p53-dependent lele #3 had a 16-nt out-of-frame deletion in exon 9. Using ferroptosis. these genetically defined HCT116 cells, we found that FDXR deficiency led to increased expression of IRP2 and The signal from FDXR to iron homeostasis and the p53 TfR1 but decreased expression of IRP1 and FTH1 (Fig. pathway is transduced by FDX2 3C). To confirm the observation that Fdxr deficiency leads to iron accumulation in developing embryos (Fig. 1), Both FDX1 and FDX2 are bona fide FDXR substrates QuantiChrom iron assay was performed and showed (Lange et al. 2000; Sheftel et al. 2010). To determine which that FDXR deficiency markedly increased the level of substrate is involved in iron homeostasis and the p53 iron in mitochondria but not in cytoplasm in MEFs and pathway, multiple FDX1- and FDX2-deficient HCT116 HCT116 cells (Fig. 3D,E). cell lines were generated. FDX1−/− clones #18 and #19 p53 expression and activity are often regulated by its had a 32-nt out-of-frame deletion in exon 1 and were cho- own targets (Jin and Levine 2001; Brooks and Gu 2011; sen for further studies. However, we were able to generate Zhang et al. 2011) and thus possibly by FDXR. To test only FDX2+/− but not FDX2−/− clones, suggesting that, this, we examined the levels of p53 and p21 proteins and like FDXR, FDX2 may be required for cell survival. found that p53 expression was inhibited by FDXR defi- FDX2+/− clones #44 and #47 had a 19-nt out-of-frame dele- ciency along with decreased expression of p21 in FDXR- tion in exon 1 and were chosen for further studies. We deficient liver tissues and cells (Fig. 3A–C; Supplemental found that, like FDXR deficiency, FDX2 deficiency led Fig. S3A–C). To confirm the role of FDXR in mitochondri- to increased expression of IRP2 and TfR1 but decreased al iron homeostasis and the p53 pathway, we examined expression of IRP1 and FTH1 (Fig. 4A). Similar results whether ectopic expression of FDXR would restore nor- were detected in HCT116 cells in which FDX2 was mal iron metabolism and p53 expression. Indeed, we knocked down by two separate siRNAs (Supplemental found that, in FDXR+/−/− cells, the levels of IRP2, TfR1, Fig. S5). In addition, FDX2 deficiency led to decreased ex- FTH1, p53, and p21 were restored to near normal by ectop- pression of p53 and p21 (Fig. 4A) as well as mitochondrial ic expression of FDXR (Fig. 3F, cf. lanes 2,3 and 5,6, respec- iron overload (Fig. 4B). In contrast, knockout or transient tively). Interestingly, IRP1 was induced by FDXR (Fig. 3F), knockdown of FDX1 had no effect on the level of IRP1, potentially due to increased association of IRP1 with Fe-S IRP2, p53, and p21 and slightly increased the level of clusters, which enhances IRP1 stability (Hentze et al. TfR1 but significantly decreased the level of FTH1 (Sup- 2010). However, in isogenic control cells, ectopic expres- plemental Fig. S6A,B). Moreover, FDX1 knockout had sion of FDXR slightly suppressed IRP2 and TfR1 expres- no effect on iron homeostasis in HCT116 cells (Supple- sion but had little if any effect on IRP1 (Fig. 3F, cf. lanes mental Fig. S6C). 1 and 4). Additionally, p53, p21, and FTH1 were induced by FDXR (Fig. 3F, cf. lanes 1 and 4). Since FTH1 is a puta- FDXR regulates iron homeostasis and p53 expression tive p53 target (Funauchi et al. 2015), it is likely that the through IRP2 induction of p21 and FTH1 is due to increased expression of p53 (Fig. 3F, cf. lanes 1 and 4). Moreover, we found that IRP2 is a master regulator of key genes in cellular iron me- mitochondrial iron overload in FDXR+/−/− cells (both tabolism, including TfR1 and FTH1 (Butt et al. 1996; Hen- clones #4 and #5) was eliminated by ectopic expression derson et al. 1996). Since FDXR deficiency induces IRP2 of FDXR (Fig. 3G). In contrast, in isogenic control cells, expression and mitochondrial iron overload (Fig. 3), we FDXR had no effect on iron homeostasis (Fig. 3G). postulate that IRP2 may mediate FDXR-dependent iron As a major regulator of iron homeostasis and a mediator homeostasis. To test this, multiple IRP2 knockout of ROS-mediated apoptosis (Hwang et al. 2001; Liu and HCT116 cell lines were generated. Clones #67 and #130, Chen 2002), FDXR may play a role in p53-dependent fer- which had a 25-nt and 44-nt out-of-frame deletion in roptosis (Jiang et al. 2015), which is characterized by lipid exon 3, respectively, were chosen for further studies. We peroxidation, generation of lipid-based ROS, and its found that TfR1 was decreased, whereas FTH1 was in- dependency on iron (Yang and Stockwell 2016). To test creased by IRP2 knockout (Fig. 5A), consistent with previ- this, we examined whether FDXR modulates the extent ous studies (LaVaute et al. 2001; Cooperman et al. 2005; of ferroptosis induced by RSL3 and Erastin. RSL3 is a glu- Galy et al. 2005). We also found that IRP1 was accumulat- tathione peroxidase 4 inhibitor, whereas Erastin is an in- ed in IRP2 knockout cells (Fig. 5A), suggesting that the hibitor of the cysteine/glutamate antiporter, both of level of Fe-S clusters is abundant for IRP1 protein stability. which are known inducers of ferroptosis (Jiang et al. Most importantly, we found that IRP2 knockout marked- 2015). Briefly, we found that RSL3- and erastin-induced ly induced p53 expression along with its targets, p21 and ferroptosis was inhibited by FDXR deficiency in both FDXR (Fig. 5B). MEFs and HCT116 cells (Supplemental Fig. S4A–H). Sur- To confirm the above observation, we generated multi- prisingly, we found that RSL- and Erastin-induced ferrop- ple IRP2−/−;FDXR+/−/− HCT116 cell lines in which both tosis was also inhibited by ectopic expression of FDXR in alleles of the IRP2 gene were deleted (44-nt deletion in HCT116 cells (Supplemental Fig. S4I–L). Consistent with exon 3), whereas two of the three alleles of the FDXR these observations, cell proliferation and colony forma- gene were deleted (20-nt deletion in one allele and 16-nt tion were increased by FDXR deficiency in MEFs and deletion in the other allele). Thus, clones #49 and #144

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Figure 4. FDX2 transduces FDXR signals to regulate iron homeostasis and the p53 pathway. (A) FDX2 regulates iron regulatory proteins and the p53 pathway. Cell lysates were collected from isogenic control HCT116 cells and FDX2+/− #44/#47 HCT116 cells and subjected to Western blot analysis with various antibodies as indicated. (B) FDX2 deficiency leads to mitochondrial iron overload. The level of cytosolic and mitochondrial iron was measured in isogenic control and FDX2+/− #44/#47 cells. (First left column) The level of cytosolic iron in isogenic control cells was set at 1.0. Data are mean ± SD from three independent experiments.

were selected for further studies. We showed that in As an RNA-binding protein, IRP2 may bind to p53 IRP2−/−;FDXR+/−/− cells, IRP2 knockout was still capable mRNA and then regulate p53 expression. To test this, of increasing expression of IRP1, FTH1, p53, and p21 IRP2 immunoprecipitation (IRP2-IP) was performed and but decreasing expression of TfR1 (Fig. 5C,D). We also showed that the p53 transcript was highly enriched in showed that IRP2 knockout had no effect on intracellu- IRP2 immunoprecipitates (Fig. 6E). As a control, TfR1 lar iron content, consistent with a recent study (Cloo- and FTH1 mRNAs, both of which are known to be recog- nan et al. 2016). However, IRP2 knockout reversed nized by IRP2 (Butt et al. 1996; Henderson et al. 1996), mitochondrial iron overload by FDXR deficiency to near were also enriched in IRP2 immunoprecipitates (Fig. normal in IRP2−/−;FDXR+/−/− cells, suggesting that IRP2 6E). To identify a region in p53 mRNA that is responsive is an important mediator acting downstream from FDXR to IRP2, multiple reporters were generated (shown in Sup- (Fig. 5E). plemental Fig. S8C). We found that in p53-null H1299 Next, we determined whether increased expression of cells, ectopic expression of IRP2 inhibited p53 expression p53 is responsible for induction of p21 and FDXR in from a vector that carries a p53-coding region plus its 3′ IRP2 knockout cells. We found that, upon knockdown untranslated region (UTR) (Fig. 6F, cf. lanes 5 and 6). How- of p53, p21 and FDXR were not induced in IRP2−/− cells ever, IRP2 had no effect on p53 expression from a reporter (Supplemental Fig. S7A). In contrast, IRP2 knockout was that carries a p53-coding region alone or together with the still capable of inducing IRP1 and FTH1 and repressing p53 5′ UTR in p53-null H1299 cells (Fig. 6F, lanes 1–4). TfR1 expression (Supplemental Fig. S7B). We note that Conversely, we found that in HCT116 cells, IRP2 knock- as a putative target of p53 (Funauchi et al. 2015), FTH1 out increased GFP expression from a reporter that carries was reduced by p53 knockdown but still induced by a GFP-coding region plus the p53 3′ UTR (Fig. 6G, lanes IRP2 knockout independent of p53 (Supplemental Fig. 5,6). However, IRP2 knockout had no effect on GFP ex- S7B). As a control, we found that knockdown of p53 led pression from a reporter that carries a GFP-coding region to decreased expression of p21 and FDXR in control alone or together with the p53 5′ UTR in HCT116 cells HCT116 cells (Supplemental Fig. S7A). (Fig. 6G, lanes 1–4). Additionally, we showed that in HCT116 cells, endogenous p53 expression was induced by IRP2 knockout regardless of the transfection with a p53 expression is regulated by IRP2 via mRNA ′ GFP reporter (Fig. 6G). Since the p53 3 UTR is responsive translation to IRP2 (Fig. 6F,G), we searched for a consensus iron-re- p53 expression is subject to transcriptional and post-tran- sponsive element (IRE) (Butt et al. 1996; Henderson scriptional regulations (Zhang and Chen 2008; Vousden et al. 1996). Indeed, we found such a conserved IRE and Prives 2009). Thus, semiquantitative and quantitative (CAGUGU) located at nucleotide 2344 in the p53 3′ RT–PCR were performed and showed that the level of p53 UTR (Supplemental Fig. S8C). Thus, we generated a transcript was not changed in FDXR+/−/− HCT116 cells GFP reporter that carries the p53 3′ UTR but without (Fig. 6A,B), FDXR knockdown HCT116 cells (Supplemen- such a conserved IRE (Supplemental Fig. S8C). Since a tal Fig. S8A), or FDXR+/− MEFs (Supplemental Fig. S8B). poly(U) region is recognized by Rbm38 to regulate p53 Similarly, the level of p53 transcript was not altered in mRNA translation (Zhang et al. 2011), we generated a IRP2−/− HCT116 cells (Fig. 6C,D). As a control, the levels GFP reporter that carries the p53 3′ UTR but without of FDXR and p21 transcripts were found to be decreased in the poly(U) (Supplemental Fig. S8C). We showed that in − − FDXR-deficient cells (Fig. 6A,B; Supplemental Fig. S8A,B) IRP2 / HCT116 cells, ectopic expression of IRP2 inhib- but increased in IRP2−/− cells (Fig. 6C,D). These results ited GFP expression from a reporter that carries an intact indicate that IRP2 may regulate p53 expression through or poly(U)-deleted p53 3′ UTR (Supplemental Fig. S8D, cf. a translational mechanism. lanes 3,7 and 4,8, respectively). In contrast, ectopic

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The p53–FDXR loop in tumor suppression

Since the binding of eIF4E to the mRNA 5′ cap is a rate- limiting step for mRNA translation, we examined whether the binding of eIF4E to the p53 5′ cap is modulated by IRP2. Indeed, we found that in HCT116 cells, the binding of eIF4E to p53 mRNA was suppressed by ectopic expression of IRP2 (Fig. 6I, cf. lanes 5 and 6) but increased by IRP2 knockout (Fig. 6J, cf. lanes 5 and 6). Since FTH1 mRNA translation is regulated by IRP2 (Gray and Hentze 1994), the level of FTH1 mRNA associated with eIF4E was examined as a control and found to be decreased by ectopic expression of IRP2 but increased by IRP2 knockout (Fig. 6I,J, cf. lanes 5 and 6).

p53 plays a role in iron homeostasis and is required for FDXR-mediated iron metabolism Since FDXR deficiency leads to decreased p53 expression along with mitochondrial iron overload (Fig. 3), we hy- pothesized that p53 plays a role in iron homeostasis. In- deed, we found that in MEFs, p53 deficiency led to increased expression of IRP2 and TfR1 but decreased expression of IRP1 and FTH1 (Fig. 7A). As a control, the levels of FDXR and p21 were decreased in p53-deficient MEFs, as expected (Fig. 7A). We also found that, like FDXR deficiency, p53 deficiency led to mitochondrial iron overload in MEFs (Fig. 7B) and iron overload in E8.0 p53−/− embryos (Fig. 7C). Additionally, iron overload was detected in Fdxr+/−, p53+/−, Fdxr+/−;p53+/−, and Fdxr+/−;p53−/− embryos (Supplemental Fig. S9A). We note that, like Fdxr+/− embryos, p53−/− embryos can toler- ate a moderate increase of iron, since p53−/− embryos are known to develop normally (Donehower et al. 1992). Next, the role of p53 in iron homeostasis was examined in HCT116 cells in which the p53 gene was deleted by CRISPR–cas9 (Fig. 7D) or knocked down by p53 siRNA (Supplemental Fig. S9B). We found that p53 deficiency led to increased expression of IRP2 and TfR1 but decreased expression of IRP1 and FTH1 in HCT116 cells Figure 5. FDXR regulates iron homeostasis and the p53 pathway (Fig. 7D; Supplemental Fig. S9B), consistent with the through IRP2. (A–D) Iron regulatory proteins and the p53 pathway above observation in p53−/− MEFs. As a control, we found are regulated by IRP2. Western blots were prepared with lysates that p53 deficiency led to decreased expression of p21 and from isogenic control HCT116 cells (A–D), IRP2−/− cells (A,B), −/− +/−/− FDXR (Fig. 7D; Supplemental Fig. S9B). Additionally, we and IRP2 ;FDXR cells (C,D) and then probed with antibod- found that p53 deficiency led to mitochondrial iron over- ies against IRP1, IRP2, FDXR, TfR1, FTH1, p53, p21, and actin, load in HCT116 cells (Fig. 7E; Supplemental Fig. S9C), respectively. (E) IRP2 knockout abolishes mitochondrial iron −/− overload in FDXR-deficient cells. The levels of cytosolic and mi- consistent with the observation in p53 MEFs. tochondrial iron were measured in isogenic control, IRP2−/−, The above observations prompted us to hypothesize FDXR+/−/−, and IRP2−/−;FDXR+/−/− HCT116 cells. (First left col- that loss of p53 plays a role in mitochondrial iron overload umn) The level of cytosolic iron in isogenic control cells was set in FDXR-deficient cells. To test this, p53 was ectopically − − at 1.0. Data are mean ± SD from three independent experiments. expressed in wild-type and FDXR+/ / HCT116 cells. As expected, p21 and FDXR were induced by ectopic expression of p53 in both wild-type and FDXR+/−/− cells (Fig. 7F). expression of IRP2 had a weakened effect on GFP expres- Interestingly, we found that the levels of IRP1 and FTH1, sion from a reporter that contains an IRE-deleted p53 3′ both of which were markedly decreased by FDXR defi- − − UTR in IRP2 / HCT116 cells (Supplemental Fig. S8D, ciency (Fig. 7F, cf. lanes 1 and 2), were increased to near cf. lanes 5 and 6). To further confirm the regulation of normal by ectopic expression of p53 (Fig. 7F, cf. lanes 2 p53 mRNA translation by IRP2, we measured the newly and 4). Similarly, the levels of IRP2 and TfR1, both of synthesized 35S-labeled p53 protein in control and IRP2 which were increased by FDXR deficiency (Fig. 7F, cf. knockout HCT116 cells. We found that the level of de lanes 1 and 2), were reduced to near normal by ectopic ex- novo synthesized p53 protein was highly increased by pression of p53 (Fig. 7F, cf. lanes 2 and 4). Furthermore, we IRP2 knockout (Fig. 6H). found that mitochondrial iron overload by FDXR

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Figure 6. p53 is regulated by IRP2 via mRNA translation. (A,B) FDXR deficiency has no effect on the level of p53 transcript. Semiquan- titative (A) and quantitative (B)RT–PCR were performed to measure the levels of FDXR, p53, p21, and actin transcripts in isogenic control and FDXR+/−/− #4/#5 cells. (C,D) IRP2 deficiency has no effect on the level of p53 transcript. Semiquantitative (C) and quantitative (D) RT–PCR were performed to measure the levels of IRP2, FDXR, p53, p21, and actin transcripts in isogenic control and IRP2−/− #67/ #130 cells. (E) RNA immunoprecipitation (RNA-IP) was performed with DNAase I-treated extracts from HCT116 cells that were transfected with control pcDNA3 (−) or a vector expressing HA-tagged IRP2 (+) for 24 h. Total RNAs were purified from IRP2 immunocomplexes and subjected to RT–PCR to measure the levels of p53, TfR1, FTH1, and actin mRNAs. (F) p53-null H1299 cells were transfected with control pcDNA3 (−) or a vector expressing HA-IRP2 (+) along with a reporter that contains the p53-coding region alone or together with the p53 5′ or 3′ untranslated region (UTR). Twenty-four hours after transfection, cell lysates were collected and subjected to Western blot analysis to detect HA-IRP2, p53, and actin. (Lane 1) The level of p53 protein in H1299 cells transfected with control pcDNA3 and a reporter that contains the p53-coding region alone was set at 1.0. (G) Isogenic control (−) and IRP2 knockout (+) HCT116 cells were transfected with a reporter that contains the GFP-coding region alone or together with the p53 5′ or 3′ UTR. Twen- ty-four hours after transfection, cell lysates were collected and subjected to Western blot analysis to detect IRP2, GFP, p53, and actin. (Lane 1) The level of GFP protein in isogenic control cells transfected with a reporter that contains GFP coding alone was set at 1.0. (H) Isogenic control and IRP2−/− HCT116 cells were labeled with 35S-methionine for 10 min. Cell lysates were isolated and used for immunoprecipitation with 1.0 µg of anti-p53. The immunoprecipitates were separated on 8% SDS-PAGE. p53 was visualized by autoradiography. The relative level of p53 was measured by densitometry. (I) HCT116 cells were transfected with control pcDNA3 (−) or a vector expressing HA- IRP2 (+) for 24 h. Using a control IgG, anti-HA that recognizes IRP2, or anti-eIF4E, RNA-IP was performed with DNAaseI-treated extracts from HCT116 cells transfected with pcDNA3 or HA-IRP2-expressing pcDNA3. Total RNAs were purified from immunocomplexes and subjected to RT–PCR to measure the levels of p53, FTH1, and actin mRNAs. (J) RNA-IP was performed with DNAase I-treated extracts from isogenic control (−) and IRP2−/− (+) HCT116 cells with control IgG or anti-eIF4E. Total RNAs were purified from immunocomplexes and subjected to RT–PCR to measure the levels of p53, FTH1, and actin mRNAs.

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Figure 7. Mitochondrial iron homeostasis is regulated by p53, and FDXR-mediated iron metabolism is p53-dependent. (A) Iron regulatory proteins are regulated by p53 in MEFs. Western blots were prepared using extracts from wild-type, Fdxr+/−, and p53−/− MEFs. The blots were probed with antibodies against FDXR, p53, p21, IRP1, IRP2, TfR1, FTH1, and actin, respectively. (B) Loss of p53 leads to mitochondrial iron overload in MEFs. The levels of cytosolic and mitochondrial iron were measured in wild- type, Fdxr+/−, and p53−/− MEFs. (First left column) The level of cytosolic iron in wild- type MEFs was set at 1.0. Data are mean ± SD from three independent experiments. (C) Loss of p53 leads to a mild iron overload in E8.0 embryos. Prussian blue staining was performed with wild-type, Fdxr+/−, and p53−/− embryos. The right panels represent the boxed regions in the left panels of each set at higher magnification. Blue staining represents iron accumulation. (D) Iron regulatory proteins are regulated by p53 in HCT116 cells. Western blots were prepared using extracts from isogenic control, FDXR+/−/−, and p53−/− HCT116 cells and probed with various antibodies. (E) Loss of p53 leads to mitochondrial iron overload in HCT116 cells. The levels of cytosolic and mitochondrial iron were measured in isogenic control, FDXR+/−/−, and p53−/− HCT116 cells. (First left column) The level of cytosolic iron in isogenic control cells was set at 1.0. Data are mean ± SD from three independent experiments. (F) Ectopic expression of p53 restores near-normal expression of iron regulatory proteins in FDXR+/−/− HCT116 cells. Isogenic control and FDXR+/−/− HCT116 cells were transfected with control pcDNA3 (−) or a vector expressing p53 (+). Cell lysates were collected and subjected to Western blot analysis with various antibodies as indicated. (G) Ec- topic expression of p53 restores near-normal iron metabolism in FDXR+/−/− HCT116 cells. The levels of cytosolic and mitochondrial iron were measured in isogenic control and FDXR+/−/− HCT116 cells transfected with control pcDNA3 (−) or a vector expressing HA-tagged p53 (+). (First left column) The level of cytosolic iron in isogenic control HCT116 cells transfected with control pcDNA3 was set at 1.0. Data are mean ± SD from three independent experiments. (H) A model of the FDXR–IRP2–p53 loop in iron homeostasis and tumor suppression.

deficiency was restored to near normal by ectopic expres- abnormalities. (3) FDXR deficiency leads to aberrant sion of p53 (Fig. 7G, last two columns). Together, our re- iron metabolism and increased expression of IRP2 that results suggest that p53 plays a role in iron homeostasis presses p53 mRNA translation. (4) p53 plays a role in iron and mediates FDXR-dependent iron metabolism. homeostasis and mediates FDXR-dependent iron metabolism. The hypothesis is summarized in Figure 7H.

Discussion The FDXR–p53 loop and mitochondrial iron overload This study showed that FDXR and p53 are mutually regu- Loss of FDXR leads to mitochondrial iron overload, which lated and that the FDXR–p53 loop is critical for tumor is likely responsible for embryonic lethality (Fig. 1B). Our suppression via iron homeostasis based on the following observation is consistent with previous studies that dis- evidence: (1) Loss of Fdxr leads to embryonic lethality ruption of genes related to iron metabolism leads to em- and cell death. (2) Mice heterozygous in Fdxr have a short bryonic lethality at E8.5–E12.5 due to oxidative stress life span and are prone to spontaneous tumors and liver (Donovan et al. 2005; Mao et al. 2010; Moroishi et al.

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2011; Ruiz et al. 2013). We also found that p53 plays a role the presence and absence of FDXR (Fig. 5; Supplemental in iron homeostasis and is required for FDXR-mediated Fig. S8). Considering that IRP2 is markedly induced along iron metabolism (Fig. 7). Thus, our findings strengthen with decreased p53 expression by FDXR deficiency, we the idea that p53 is a critical modulator of mitochondrial conclude that p53 expression is regulated primarily by integrity and energy and iron metabolism (Zhou et al. IRP2 in FDXR-deficient cells. Additionally, we found 2003; Matoba et al. 2006; Funauchi et al. 2015). that ectopic expression of IRP2 inhibits, whereas loss of Mitochondrial iron overload is observed in a number of IRP2 increases, the association of eIF4E with the p53 5′ diseases, including sideroblastic anemia and cancer. Mito- cap (Fig. 6I,J), indicating that IRP2 represses p53 mRNA chondrial iron overload can be induced by increased im- translation in a cap-dependent manner. These results port, decreased export, or both, but it remains unclear are consistent with a previous report that the binding what drives mitochondrial iron accumulation (Huang of IRP2 with an IRE on its target mRNA prevents the as- et al. 2011; Rouault 2016). Import of iron to mitochondria sembly of the 43S translation preinitiation complex is controlled primarily by inner mitochondrial matrix pro- (Gray and Hentze 1994). Generally, IRP2 recognizes an teins: mitoferrin 1 and mitoferrin 2. Indeed, mitoferrin ex- IRE located in the 5′ UTR for mRNA translation and pression is found to be altered in FDX1/2 knockdown cells an IRE in the 3′ UTR for mRNA stability (Butt et al. (Shi et al. 2012). While mitoferrin is regulated primarily by 1996; Henderson et al. 1996). Interestingly, we found transcription, it is not known what controls mitoferrin that a putative IRE in the p53 3′ UTR is responsive to transcription (Rouault 2016). Iron export is known to IRP2 (Fig. 5; Supplemental Fig. S8). We note that the be controlled by ABCB7, an ABC transporter in the mito- p53 3′ UTR is recognized by and responsive to RBM38 chondrial matrix and a potential Fe-S exporter. Inter- and RPL26 for translational control (Takagi et al. 2005; estingly, IRP2 knockout reverses mitochondrial iron Zhang et al. 2011). Thus, we hypothesize that p53 overload by FDXR deficiency to near normal (Fig. 5). Addi- mRNA translation is uniquely regulated through its 3′ tionally, loss of FDXR leads to increased expression of UTR by multiple RNA-binding proteins. Nevertheless, IRP2, which suppresses p53 expression (Figs. 3–5). Thus, it remains possible that IRP1, which is likely to be regu- our data suggest that IRP2 has a novel activity as a mito- lated by FDXR, may modulate p53 translation through chondrial iron regulator. Moreover, mitochondrial mem- binding to the p53 5′ UTR. brane potential, which is known to be regulated by FDXR and p53 via ROS production (Li et al. 1999; Charlot FDXR deficiency and tumorigenesis et al. 2004), may play a role in mitochondrial iron import and export. Together, we speculated that IRP2 and p53 co- Mice deficient in Fdxr are prone to spontaneous tumors ordinately regulate these critical iron importers and ex- and liver abnormalities (Fig. 2; Supplemental Fig. S2; Sup- porters through transcription, RNA stability, and/or plemental Table S2). Since p53 expression is suppressed translation. by FDXR deficiency, we postulate that decreased expression of p53 is a major contributing factor. However, compared with p53-deficient mice (Donehower and Lozano FDXR, a potential mediator of p53-dependent ferroptosis 2009), we found that mice deficient in Fdxr are prone to Previously, we and others showed that FDXR is a media- a broader spectrum of tumors, including lung adenocarci- tor of p53-dependent ROS-induced apoptosis (Hwang noma and HCC (Fig. 2; Supplemental Fig. S2; Supplemen- et al. 2001; Liu and Chen 2002) and modulates Fhit-medi- tal Tables S1, S2), suggesting that other pathways altered ated apoptosis (Trapasso et al. 2008). Additionally, by Fdxr deficiency may play a role in tumorigenesis. First, through interaction with MPZL, FDXR plays a role in we hypothesize that mitochondrial iron overload leads to ROS-induced epidermal cell differentiation via mitochon- increased liver inflammation (hepatitis), which would dria (Bhaduri et al. 2015). Here, we found that, by modu- synergize with loss of p53 to promote cell transformation. lating RSL3- and Erastin-induced ferroptosis, FDXR Second, FDXR is a key regulator of steroidogenesis in mi- plays a role in p53-dependent ferroptosis (Supplemental tochondria (Muller et al. 2001). Thus, Fdxr deficiency Fig. S4). Interestingly, RSL3- and Erastin-induced ferrop- would alter lipid metabolism, leading to liver steatosis tosis are suppressed by FDXR deficiency as well as as observed in Fdxr-deficient mice (Fig. 2; Supplemental FDXR overexpression. As a common target of the p53 Fig. S2; Supplemental Table S2). Since liver steatosis pre- family (Liu and Chen 2002) and a mediator of p53-depen- disposes livers to hyperplasia and eventually HCC (Mey- dent apoptosis and ferroptosis, further studies are war- nard et al. 2014), we hypothesize that liver steatosis ranted to determine the mechanism by which FDXR would synergize with loss of p53 to promote cell (hepato- modulates ferroptosis, whether p63 and p73 are capable cyte) transformation. Third, FDXR is known to interact of inducing ferroptosis via FDXR, and how FDXR coordi- with Fhit and may play a role in Fhit-mediated tumor sup- nately regulates p53-dependent ROS-mediated apoptosis pression (Trapasso et al. 2008). Thus, we hypothesize that and ferroptosis. loss of p53 in FDXR-deficient cells may synergize with loss of Fhit-mediated tumor suppression to promote tumorigenesis. Fourth, increased expression of IRP2 in IRP2 and p53 mRNA translation FDXR-deficient cells can promote c-Myc expression (Wu We found that ectopic expression of IRP2 inhibits, et al. 1999; Maffettone et al. 2010), which would cooperate whereas knockout of IRP2 increases, p53 expression in with loss of p53 to promote tumorigenesis.

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FDX2—the primary transducer of FDXR for iron were approved by the University of California at Davis Institu- homeostasis and tumor suppression tional Animal Care and Use Committee. Both FDX1 and FDX2 can carry out biogenesis of Fe-S clusters (Shi et al. 2012), but FDX2 is necessary and suffi- Cell culture cient for such an activity (Sheftel et al. 2010). Here, we HCT116, HepG2, H1299, and their derivatives were cultured in showed that FDX1 is not required for cell survival and DMEM (Dulbecco’s modified Eagle’s medium, Invitrogen) sup- has no effect on iron homeostasis and p53 expression (Sup- plemented with 10% fetal bovine serum (Hyclone). Wild-type plemental Fig. S6). Since FDX1 is necessary and sufficient and Fdxr+/− MEFs were generated as described previously (Zhang for steroidogenesis in mitochondria (Sheftel et al. 2010), et al. 2011) and cultured in DMEM supplemented with 10% fetal we hypothesize that loss of the signal from FDXR to bovine serum, 55 µM β-mercaptoethanol, and MEM nonessential FDX1 is likely responsible for liver steatosis in Fdxr-defi- amino acid solution (Cellgro). cient mice (Fig. 2; Supplemental Fig. S2; Supplemental Ta- ble S2). In contrast, FDX2 is required for cell survival, and Plasmid construction and cell line generation FDX2 deficiency leads to mitochondrial iron overload and FDXR, FDX1, FDX2, IRP2, and p53 guide RNAs (gRNAs) were de- decreased p53 expression. Additionally, FDX2 deficiency signed using CRISPR design tool (http://crispr.mit.edu) and are leads to increased IRP2 expression (Fig. 4A; Supplemental listed in Supplemental Table S3. To generate a vector expressing Fig. S5). These data indicate that FDX2 is the primary sub- a single gRNA (sgRNA), two 25-nt oligos were annealed and then strate to transduce the FDXR signal via IRP2 to iron ho- cloned into the pSpCas9(BB) (Ran et al. 2013) sgRNA expression meostasis, p53 expression, and tumor suppression. vector. To generate a knockout cell line, HCT116 cells were transfected with a gRNA-expressing vector and selected with pu- romycin. Cells deficient in FDXR, FDX1, FDX2, IRP2,orp53 Conclusion and future direction were confirmed by genotyping, sequencing, and Western blot analysis. The primers used for sequencing FDXR, FDX1, FDX2, Our data together with published studies indicate that the IRP2, and p53 are listed in Supplemental Table S4. FDXR–p53 loop plays a critical role in iron homeostasis pcDNA3 vectors carrying a p53- or GFP-coding region alone or for tumor suppression. As a target of the p53 family, we together with the p53 5′ or 3′ UTR (Supplemental Fig. S8C) were speculate that FDXR may engage other p53 family mem- used as described previously (Zhang et al. 2013). GFP reporters ′ bers (that is, p63 and p73) to regulate iron homeostasis. that carry a p53 3 UTR with deletion of the putative IRE or Importantly, we speculate that FDXR would modulate poly(U) region were generated by two-step PCR reactions (Sup- mutant p53 gain of function through iron metabolism. plemental Fig. S8C). The pcDNA4 vector expressing HA-tagged FDXR was generated by subcloning the HA-FDXR from pUHD Indeed, a recent study showed that FDXR knockdown 10-3-HA-FDXR (Liu and Chen 2002) to the pcDNA4 vector via suppresses T47D mammary tumor cell growth and xeno- EcoRI. The primers are listed in Supplemental Table S5. graft (Zhang et al. 2015), potentially due to decreased expression of mutant p53 (L194F) in T47D cells. Thus, further studies are warranted to determine whether iron Western blot analysis homeostasis is regulated by other p53 family pathways Western blot was performed as described (Zhang et al. 2014). An- via FDXR and whether the FDXR–p53 loop can be ex- tibodies against p53, p21, IRP2, and TfR1 were purchased from plored for managing tumors that carry wild-type p53 as Santa Cruz Biotechnology. Anti-mouse p53 (1C12) was purchased well as tumors addicted to mutant p53. from Cell Signaling Technology. Antibodies against FDXR, FDX1, FDX2, IRP1, and FTH1 were purchased from Abcam. Anti-HA was purchased from Covance. Anti-actin and HRP-con- Materials and methods jugated secondary antibodies against rabbit or mouse IgG were purchased from Bio-Rad. The immunoreactive bands were visual- Reagents ized by enhanced chemoluminescence (Thermo Fisher Scientific, Inc.) and quantified by densitometry with the BioSpectrum 810 Proteinase inhibitor cocktail, RNase A, and protein A/G beads imaging system (UVP LLC). were purchased from Sigma. Potassium ferrocyanide, nuclear Fast Red, and hematoxylin and eosin (H&E) were purchased from Fisher Scientific. RNA isolation and RT–PCR analysis Total RNAs were extracted from cells using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s instructions. Fdxr- and p53-deficienct mouse models cDNA was synthesized using M-MLV reverse transcriptase kit Fdxr+/− mice were generated by the Mouse Biology Program at (Promega Corporation) according to the manufacturer’s protocol. University of California at Davis. The targeting vector carries The levels of p53, p21, FDXR, and actin transcripts were mea- the entire Fdxr locus in which exons 1–12 were replaced by sured by semiquantitative and/or quantitative PCR with primers LacZ-poly(A) and hUbDpro-neo-poly(A) cassettes. Mutant em- listed in Supplemental Tables S6 and S7. bryonic stem cells were microinjected into C57BL/6Ntac blasto- cytes to generate chimeras. The resulting chimeras were crossed RNAi with C57BL/6 mice for germline transmission, and the offspring were backcrossed with C57BL/6 mice for more than six genera- Scrambled siRNA and siRNAs against FDXR (siFDXR#1 and tions. The p53+/− mice (on a C57BL/6 background) were pur- siFDXR#2), FDX2 (siFDX2#1 and siFDX2#2), and p53 (sip53) chased from the Jackson Laboratory. All animal procedures were purchased from Dharmacon and are listed in Supplemental

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Table S8. For siRNA transfection, siLentFectTM lipid reagent References (Bio-Rad) was used according to the user’s manual. Abend M, Badie C, Quintens R, Kriehuber R, Manning G, Mac- aeva E, Njima M, Oskamp D, Strunz S, Moertl S, et al. 2016. Histological analysis, iron histochemistry, and reticulin staining Examining radiation-induced in vivo and in vitro gene expression changes of the peripheral blood in different laboratories Mouse tissues or embryos were fixed in 10% (w/v) neutral buff- for biodosimetry purposes: first RENEB gene expression study. ered formalin, processed, and embedded in paraffin blocks. Em- Radiat Res 185: 109–123. bedded tissues were sectioned (5 µm) and stained with H&E. 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Transcript analysis for internal biodosimetry edited the manuscript. using peripheral blood from neuroblastoma patients treated

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Ferredoxin reductase is critical for p53-dependent tumor suppression via iron regulatory protein 2

Yanhong Zhang, Yingjuan Qian, Jin Zhang, et al.

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